Field of the Invention
The present invention relates to fermentation processes, to microorganisms and to substrates useful for fermentation. In particular, this invention is related to the production of 1,2-propanediol by fermentation, from a sucrose-containing medium, in particular from plant biomass.
Description of Related Art
1,2-propanediol or propylene glycol, a C3 dialcohol, is a widely-used chemical. It is a component of unsaturated polyester resins, liquid detergents, coolants, anti-freeze and de-icing fluids for aircraft. Propylene glycol has been increasingly used since 1993-1994 as a replacement for ethylene derivatives, which are recognised as being more toxic than propylene derivatives.
1,2-propanediol is currently produced by chemical means using a propylene oxide hydration process that consumes large amounts of water. Propylene oxide can be produced by either of two processes, one using epichlorhydrin, and the other hydroperoxide. Both routes use highly toxic substances. In addition, the hydroperoxide route generates by-products such as tert-butanol and I-phenyl ethanol. For the production of propylene to be profitable, a use must be found for these by-products. The chemical route generally produces racemic 1,2-propanediol, whereas each of the two stereoisomers (R)1,2-propanediol and (S)1,2-propanediol are of interest for certain applications (e.g. chiral starting materials for specialty chemicals and pharmaceutical products).
The disadvantages of the chemical processes for the production of 1,2-propanediol make biological synthesis an attractive alternative. Two routes have been characterized for the natural production of 1,2-propanediol from sugars by microorganisms. In the first route 6-deoxy sugars (e.g. L-rhamnose or L-fucose) are cleaved into dihydroxyacetone phosphate and (S)-lactaldehyde, which can be further reduced to (S)-1,2-propanediol (Badia et al, 1985). This route is functional in E. coli, but cannot yield an economically feasible process due to the elevated cost of the deoxyhexoses. The second route is the metabolism of common sugars (e.g. glucose or xylose) through the glycolysis pathway followed by the methylglyoxal pathway. Dihydroxyacetone phosphate is converted to methylglyoxal that can be reduced either to lactaldehyde or to acetol. These two compounds can then undergo a second reduction reaction yielding 1,2-propanediol. This route is used by natural producers of (R)-1,2-propanediol, such as Clostridium sphenoides and Thermoanaerobacter thermosaccharolyticum. These two organisms have been used to produce 1,2-propanediol from different sugars, namely monosaccharides (D-glucose, D-mannose, D-galactose for the hexoses and D-xylose or L-arabinose for the pentoses) or disaccharides (lactose or cellobiose) or mixtures (Tran Din and Gottschalk, 1985, Cameron and Cooney, 1986, Sanchez-Rivera et al, 1987, Altaras et al, 2001). The best performance obtained was a titer of 9 g/l and a yield from glucose of 0.2 g/g (Sanchez-Rivera et al, 1987). However, the improvement of the performances obtained with these organisms is likely to be limited due to the shortage of available genetic tools. The same synthesis pathway is functional in E. coli or other Entcrobacteriaccac and several investigations have been done by the group of Cameron (Cameron et al, 1998, Altaras and Cameron, 1999, Altaras and Cameron, 2000) and the group of Bennett (Huang et al, 1999, Berrios-Rivera et al, 2003) for the production of 1,2-propanediol in this organism with carbon sources limited to D-glucose or D-fructose. The best result obtained in an anaerobic fed-batch fermenter was a production of 4.5 g/l 1,2-propanediol with a yield of 0.19 g/g from glucose (Altaras and Cameron, 2000). Results obtained with the same approach but with lower titers and yields are also described in the patent WO 98/37204, although using different carbon sources, namely galactose, lactose, sucrose and xylose but also glucose. The titers obtained with disaccharides (lactose and sucrose) were very low (6 mg/l and 7 mg/l respectively). Better production results were described with a rationally designed then evolved E. coli strain in patent application WO 2005/073364. A 1,2-propanediol titer of 1.8 g/l was obtained, with a yield of 0.35 g/g of glucose consumed. Production of 1,2-propanediol and hydroxyacetone was also described using recombinant yeast in patent WO 99/28481.
Carbon sources used in fermentation media generally consisted in carbohydrates, mostly derived from plants. Sucrose is obtained from sugar plants such as sugar beet, sugarcane, sweet sorghum, sugar maple, sugar palms or blue agaves. Starch is the most abundant storage carbohydrate in plants. The most important starch sources are cereals (corn, wheat, rice), manioc, sweet potatoes and potatoes. Starch is not metabolized by most microorganisms but can be processed to fermentable feedstocks by the starch industry. Inulin or inulin-like polymers (mainly consisting of fructose units) are the second most abundant storage carbohydrate in plants and are found in chicory, Jerusalem artichoke or dahlia. Lignocellulosic biomass composed of cellulose, hemicellulose and lignin is also a promising source of carbohydrate but still under development (Peters, 2006). As the cost of the biotechnologically produced commodity chemicals are mainly related to the cost of raw material (i.e. the cost of the fermentation substrate), use of refined sugars such as glucose or sucrose is not an economically sustainable choice for industrial scale production. Less expensive substrates are needed that retain a high content of fermentable sugar. In this respect, sucrose containing carbon sources coming from the sugar industry represent a good option.
Sucrose is produced from sugar beet containing 16 to 24% sucrose by sugar beet processing in several steps. The cleaned and washed beets are sliced into long strips called cossettes that are extracted with hot water by diffusion to get a sucrose juice called raw juice and containing 10 to 15% sucrose. The second step is the purification of the raw juice by alkalization and carbonation using lime and then carbon dioxide to remove the impurities and get the thin juice. The evaporation process increases the sucrose concentration in the thin juice by removing water to get the thick juice with a sucrose content of 50 to 65%. This concentrated sucrose juice is then crystallised and the crystals are separated by centrifugation and then washed and dried to get pure sugar. One or more crystallisation steps can be applied to get sucrose of various purity grades. By-products of sugar beet processing include pulp (the exhausted cossettes) and molasses (the remaining mother-liquor from the crystallisation having still a sucrose content of 40 to 60%).
Sucrose is also produced from sugar cane (7 to 20% sucrose content) by the sugar industry. The harvested sugar cane is cleaned before the milling process for extraction of the juice. The structure of the cane is broken and then grinded and at the same time the sucrose is extracted with water to get the raw juice. The crushed cane exhausted from sugar is called bagasse. This residue is primarily used as fuel source to generate process steam. The raw juice is then clarified by adding lime and heating and the clarified juice is separated from the precipitate. The lasts steps of the process, evaporation to get a concentrated syrup and crystallisation are essentially the same as for the sugar beet processing. The by-products of sugarcane processing include bagasse, filter cake from clarification of raw juice and different kind of molasses, still containing significant amount of sucrose.
The different sucrose containing intermediates, products or by-products from the sugar processes (raw juice, thin or clarified juice, thick juice, sucrose syrup, pure sucrose, molasses) may serve as fermentation feedstock. For example, the sugar industry in Brazil is using the clarified sugarcane juice for ethanol production in order to use it as a substitute to gasoline. Recent examples in literature using crude sucrose containing products include ethanol production from sugar beet diffusion juice by Zymomonas mobilis (Beckers et al., 1999), production of D-lactate from molasses by E. coli (Shukla et al., 2004) and production of D-lactate from sugarcane molasses, sugarcane juice or sugar beet juice by Lactobacillus delbrueckii (Calabia et al., 2007).
Two different systems have been characterized for the uptake and utilization of sucrose in sucrose-positive bacteria (i.e. bacteria able to utilize sucrose)
The first one is based on a phosphoenol pyruvate (PEP)-dependent sucrose phosphotransferase system (sucrose PTS) where sucrose is taken up and phosphorylated using phosphoenol pyruvate (PEP) as a donor to yield intracellular sucrose-6-phosphate. Sucrose-6-phosphate is then hydrolysed to D-glucose-6-phosphate and D-fructose by an invertase. D-fructose is further phosphorylated to D-fructose-6-phosphate by an ATP-dependent fructokinase and can then enter the central metabolism. Such a system has been described in several bacterial species, gram-positive as well as gram-negative. Among Entcrobacteriaccac, more than 90% of wild-type Klebsiella but less than 50% of Escherichia and less than than 10% of Salmonella strains are sucrose positive.
A conjugative plasmid pUR400 bearing the genes scrKYABR coding for the sucrose PTS has been isolated from Salmonella (Schmid et al., 1982, Schmid et al., 1988).
A second non-PTS system was discovered more recently in E. coli EC3132 (Bockmann et al., 1992). This system involve the genes cscBKAR coding for a sucrose:proton symport transport system (CscB), a fructokinase (CscK), an invertase (CscA) and a sucrose-specific repressor (CscR).
Escherichia coli K12 and its derivatives (including MG1655) cannot utilize sucrose. However, this ability can be conferred by the transfer of the genes coding for the two previously described systems. This has been demonstrated by transferring the plasmid pUR400 in E. coli K12 (Schmid et al, 1982) or different plasmids (including pKJL101-1) bearing the cscBKAR genes in a sucrose negative strain of E. coli (Jahreis et al., 2002). As for industrial application, tryptophan production from sucrose has been documented in E. coli K12 (Tsunekawa et al., 1992), hydrogen production was shown in E. coli carrying the pUR400 plasmid (Penfold and Macaskie, 2004) and production of different amino-acids by transferring both systems, PTS and non-PTS was reported in patent application EP1149911.
Production of 1,2-propanediol from sucrose is mentioned in Clostridium thermosaccharolyticum (later renamed T. thermosaccharolyticum) by Cameron and Cooney (1986) but only traces were recorded whereas amount higher than 3 g/l with yields higher than 0.1 g/g substrate were obtained with other carbon sources.
Production of 1,2-propanediol from sucrose is also mentioned in patent application WO98/37204. However, the strain E. coli AA200 transformed with the plasmid pSEARX produces only 7 mg/l of 1,2-propanediol from 10 g/l of sucrose, whereas the same microorganism produces from 49 to 71 mg/l of 1,2-propanediol from monosaccharides. These very low figures of production cast doubt about the true ability of these organisms to produce 1,2-propanediol from sucrose. In our opinion, the strain AA200, which is derived from E. coli K12, should not have the ability to import and then metabolize sucrose.
These previous reports clearly indicated to the man skilled in the art that the use of sucrose to produce 1,2-propanediol was not a good option.
Surprisingly, by introducing different systems for sucrose utilization in E. coli strains unable to utilize sucrose, the inventors of the present invention were able to obtain improved yield for 1,2-propanediol production from sucrose.
Furthermore, the inventors demonstrated that any sucrose-containing medium, such as a juice or molasses from a plant feedstock, could be used as a substrate for the fermentative production of 1,2-propanediol.